Method of detecting an analyte in a fluid

ABSTRACT

A method for the detection of an analyte in a fluid, comprising contacting the fluid with a holographic element comprising a medium and a hologram disposed throughout the volume of the medium, wherein an optical characteristic of the element changes as a result of a variation of a physical property occurring throughout the volume of the medium, wherein the variation arises as a result of interaction between the medium and the analyte, and wherein the reaction and the variation are reversible; and detecting any change of the optical characteristic.

FIELD OF THE INVENTION

This invention relates to a method of detection based on a sensitiveelement which is a hologram, and a device for use in such a method.

BACKGROUND TO THE INVENTION

A short communication to the International Journal of Optoelectronics7(3):449452 (1992), by Spooncer et al, entitled “A humidity sensor usinga wavelength-dependent holographic filter with fibre optic links”,describes the response of gelatin-based Bragg reflection holograms toambient humidity. It concludes that optical response to an increasingand decreasing cycle of humidity shows a hysteresis which limits itsindustrial application as a sensor.

WO-A-9526499 discloses a holographic sensor, based on a volume hologram.This sensor comprises an analyte-sensitive matrix having an opticaltransducing structure disposed throughout its volume. Because of thisphysical arrangement of the transducer, the optical signal generated bythe sensor is very sensitive to volume changes or structuralrearrangements taking place in the analyte-sensitive matrix as a resultof interaction or reaction with the analyte. For example, a sensorcomprising a gelatin-based holographic medium may be used to detecttrypsin. Trypsin acts on the gelatin medium, irreversibly destroying theintegrity of the holographic support medium.

SUMMARY OF THE INVENTION

According to one aspect of the invention, a method for the continuousdetection of an analyte in a fluid, comprises contacting the fluid witha holographic element comprising a medium and a hologram disposedthroughout the volume of the medium, wherein an optical characteristicof the element changes as a result of a variation of a physical propertyoccurring throughout the volume of the medium, wherein the variationarises as a result of interaction between the medium and the analyte,and wherein the reaction and the variation are reversible; andmonitoring the optical characteristic.

The variation arises as a result of interaction between the medium andthe analyte, wherein the interaction and the variation are reversible.The interaction may be a chemical or biochemical reaction. Since boththe interaction and the reverse interaction can occur, an analytespecies can be continuously detected, preferably in real time. Theanalyte concentration may change, while the fluid is static.Alternatively, the fluid may be passed continuously over the element.

The invention may be used to monitor a reaction in vivo or in vitro,e.g. in a fermenter. It can be used for kinetic measurement, and as aneffective control system.

Another aspect of the invention is a device for the detection of ananalyte in a fluid. The device comprises a sensor comprising theholographic element, and an inlet and outlet which allow the fluid to bebrought into contact with, or passed over, the holographic element. Thedevice also includes a window for non-ionising radiation to irradiatethe holographic element. A reaction occurring in the element can thus beobserved.

DESCRIPTION OF PREFERRED EMBODIMENTS

The interaction between the holographic support medium and the analyteis reversible, and therefore continuous detection of the analyte may beachieved. When the fluid is in contact with the holographic element, theanalyte and support medium interact, preferably by a chemical orbiochemical reaction. If the fluid passes over the element, theinteraction may be transient.

The interaction can be detected remotely, using non-ionising radiation.The extent of interaction between the holographic medium and the analytespecies is reflected in the degree of change of the physical property,which is detected as a variation in an optical characteristic,preferably a shift in wavelength of non-ionising radiation.

The property of the holographic element which varies may be its chargedensity, volume, shape, density, viscosity, strength, hardness, charge,hydrophobicity, swellability, integrity, cross-link density or any otherphysical property. Variation of the or each physical property, in turn,causes a variation of an optical characteristic, such as polarisability,reflectance, refractance or absorbance of the holographic element.

The hologram may be disposed on or in, part of or throughout the bulk ofthe volume of the support medium. An illuminating source of non-ionisingradiation, for example visible light, may be used to observevariation(s) in the, or each, optical characteristic of the holographicelement.

More than one hologram may be supported on, or in, a holographicelement. Means may be provided to detect the or each variation inradiation emanating from the or each hologram, arising as a result of avariation in the or each optical characteristic. The holographicelements may be dimensioned and arranged so as to sense two independentevents/species and to affect, simultaneously, or otherwise, radiation intwo different ways. Holographic elements may be provided in the form ofan array.

Different types of hologram exist. One or more of these may be producedin, or on, the holographic support medium. Some different types ofhologram are described below.

A holographic element with the properties of a “phase” hologram maycomprise a 3-D distribution (modulation) of refractive index where thedistribution is a physical record of the original interference pattern.A holographic element with the properties of an “amplitude” hologramcomprises a 3-D distribution (modulation) of a radiation-refractingmaterial wherein the distribution is a physical record of an originalinterference pattern. Peaks of the modulation are referred to asfringes. A hologram can have the properties of a “phase” and/or an“amplitude” hologram.

The radiation may experience a phase shift as a result of modificationto the distribution of index of refraction arising from a change inspacing between peaks of a distribution supported in part, or throughoutthe volume of, the support medium. A change in fringe separation may bemeasured by peak (Bragg) wavelength change at a fixed angle ofincidence/diffraction, by monochromatic intensity change at a fixedangle, or by an angle change at monochromatic peak intensity.

Holograms can be further categorised into four distinct types which canco-exist in the same support medium. These are transmission, reflection,edge-lit and surface holograms. There are also evanescent-waveholograms.

A “transmission” hologram is one where the emergent rays leave theholographic support medium via the surface opposite to that by whichincident rays enter. Fringes are usually inclined to the surface at aconsiderable angle, e.g. typically around 900.

A “reflection” hologram is one where rays leave by the same surface atwhich incident rays enter. Fringes are usually substantially parallel tothe surface of the holographic support medium.

“Edge-lit” holograms are ones where rays leave the hologram substrate orbulk of holographic support medium (e.g. glass plate) via a surfacewhich is substantially 90° to that via which incident rays enter.Fringes are usually at an angle to the surface, typically of around 45°.

A “surface” hologram is one where the surface of a medium is contouredwith an appropriate spatial amplitude and with a regularly spacedpattern so that it is capable of diffracting and/or reflecting light.This has the properties of another type of “phase” hologram by virtue ofcreating a path difference between diffracted and/or reflected raysarriving at a common point from each point on its surface. If such asurface is defined on a transparent medium then light transmittedthrough the medium is subjected to periodic phase changes across thesurface due to the variation in optical path length imposed by therefractive index of the bulk of the medium.

The holographic support medium is one in which a hologram can be madeand which is capable of exhibiting one or more of the properties of thesensitive mechanisms described below. The support medium preferablycomprises a native or modified matrix with viscoelastic properties whichalter as a result of an interaction with an analyte species.

For example, the matrix is formed from the copolymerisation of(meth)acrylamide and/or (meth)acrylate-derived comonomers. Inparticular, the monomer HEMA (hydroxyethyl methacrylate) is readilypolymerisable and cross-linkable. PolyHEMA is a versatile supportmaterial since it is swellable, hydrophilic and widely biocompatible.

Other examples of holographic support media are gelatin, K-carageenan,agar, agarose, polyvinyl alcohol (PVA), sol-gels (as broadlyclassified), hydro-gels (as broadly classified), and acrylates. Furthermaterials are polysaccharides, proteins and proteinaceous materials,oligonucleotides, RNA, DNA, cellulose, cellulose acetate, siloxanes,polyamides, polyimides and polyacrylamides. Gelatin is a standard matrixmaterial for supporting photosensitive species, such as silver halidegrains. Gelatin can also be photo-cross-linked by chromium III ions,between carboxyl groups on gel strands. These materials may also be usedin combinations of two or more.

The polymer composition may be optimised to obtain a high quality film,suitable for the preparation of a reflection hologram. The film shouldallow for the production of a uniform matrix, in which holographicfringes can be formed.

Examples of analytes which may be identified and quantified by theinvention include gases and liquids such as ions, metabolites,antigens/antibodies, glucose, oxygen, carbon dioxide, urea, ions,including protons (for pH detection), alcohols, sulphides, and lactates.This list of analytes is given by way of example only. It will beevident that other analyte species exist and can be identified using asuitable holographic sensor, in accordance with the present invention.

The invention may be used to detect an analyte in a bodily fluid, forexample urine, blood or an optical fluid. A particular analyte ofinterest is glucose, whose levels in the eye are known to correlate withthose in the blood. The invention thus may be used to monitor bloodlevels of glucose indirectly by monitoring the levels in an opticalfluid such as tears.

There are a number of basic ways to change a physical property, and thusvary an optical characteristic. A combination of one or more of thesemay be employed to affect a change in the hologram and/or holographicsupport medium, so as to give rise to a change in a physical property ofthe holographic element. If any change occurs whilst the hologram isbeing replayed by incident broad band, non-ionising electromagneticradiation, then an optical property varies and a colour or intensitychange, for example, may be observed.

A physical property which may be varied is the modulation of complexindex of refraction. This may be changed by chemical modification of theholographic element, in order to change one or more optical properties.For example, an enzyme can initially enhance the depth of the modulationof complex index of refraction by cleaving at sites in between fringes.Preferably, the sensor includes a holographic element comprising amedium containing a spatial distribution of modulated index ofrefraction, which can be modified by the addition of an analyte species,such that the spectral and/or directional nature of incident radiationis modified in dependence upon a variation in said spatial distributionof modulated index of refraction.

The holographic element may also be prepared so that its response to aninteraction with an analyte is a temperature change. One or moredimensions of the holographic element, for example, will vary as aresult of the temperature change. This results in a change in one ormore optical properties.

The physical property that varies is preferably the size or volume ofthe support medium. This may be achieved by incorporating, into thesupport matrix, groups which undergo a reversible change uponinteraction with the analyte, and cause an expansion or contraction ofthe support medium. The support medium may comprise a polymer orcopolymer matrix, on or in which the groups are immobilised or present,e.g. in an interpenetrating network. An example of such a group is thespecific binding conjugate of an analyte species. Imprinted polymers, orsynthetic or biological receptors, may be used.

Another variation is in the active water, solvent or charge content ofthe support medium. In this case, the holographic support medium ispreferably in the form of a gel.

Analyte molecules that can react with at least two functional groups inthe element may form a reversible cross-link between separate parts ofthe support matrix, thereby altering the visco-elastic properties of thesupport matrix. Consequently, if present within a solvent-containingenvironment, and the support matrix changes, the support matrixcontracts and the separation of the fringes is reduced. Specificity maybe provided by ensuring that specific binding sites are provided withinthe gel matrix.

One parameter determining the response of such a system is the extent ofcross-linking. The number of cross-linking points due to polymerisationof monomers should not be so great that complex formation betweenpolymer and analyte-binding groups is relatively low, since the polymerfilm may become too rigid. This may inhibit the swelling of the supportmedium.

By way of example of a glucose sensor, for the continuous detection ofglucose, a hydrogel-based hologram may have a support medium comprisingpendant glucose groups and a lectin, preferably concanavalin A (con A).The lectin binds to the pendant glucose groups and acts as across-linker in the polymer structure. In the presence of freelydiffusible glucose, the extent of cross-linking will decrease as glucosein solution displaces polymer-attached glucose from the binding sites onthe lectin, resulting in swelling of the polymer. Volume changes inhydrogel films containing pendant glucose groups and con A can beobserved using a reflection hologram. A volume change in the hydrogelalters the fringe separation of the holographic structure and can befollowed as a shift in the peak wavelength of the spectral reflectedresponse.

Water-based systems are preferred in such a holographic sensor, sincethey protect the lectin from exposure to organic solvents. Examples ofsuitable glucose components are high molecular weight dextran, and themonomers allylglucoside and 2-glucosyloxyethyl methacrylate (GEMA).Dextran, having no inherent polymerisable functionality, can beentrapped during the polymerisation of acrylamide-based monomers;allylglucoside and GEMA can be polymerised either individually ortogether with comonomers. The polymers are preferably prepared as thinfilms on glass supports.

A holographic glucose sensor can be constructed using any other suitableglucose receptor which allows a reversible change in a physical propertyof the support medium upon binding with glucose. For example, thesupport medium may comprise pendant boronate groups, such asphenylboronate. Two adjacent diol groups in glucose bind with a boronategroup in a reversible condensation reaction. Thus in a holographicelement, reaction of glucose with pendant boronate groups causes anexpansion of the support medium, due to the size of glucose and itsassociated hydration shell. This expansion is observed as a shift in thereflectance maxima to longer wavelengths.

The continuous sensing of oxygen can be achieved by incorporating groupswhich reversibly bind oxygen into the holographic support medium.Example of suitable groups are complexes of a transition metal such ascobalt, nickel, iridium or ruthenium, e.g. complexes such asIr(PPh₃)₂(CO)Cl (“Vaska's complex”), tetracyanocobaltates, and porphyrinring complexes, in particular the haem proteins haemoglobin andmyoglobin. These groups can be immobilised onto the polymer matrix ofthe support medium. Upon binding with oxygen, these groups undergo aconformational shift resulting in an expansion of the hologram. Thisexpansion is detected optically as a shift in wavelength.

Similar conformational shifts occur for macrocyclic groups such as crownethers, which reversibly bind a range of ionic species. Crown ethers arewell known to reversibly bind Group I and Group II metal ions. Thereforea crown ether which is specific to an ionic analyte can be immobilisedin the support medium and used to continuously monitor the presence ofthe analyte.

The following Examples illustrate the invention, in conjunction with theaccompanying drawings.

EXAMPLE 1 Detection of Glucose

A monomer mixture of 0.36 g acrylamide, 0.42 g methacrylamide, 0.35 gN,N-dimethylacrylamide, 1 ml water, 40 μl 5% DMPA, 0.03 g methylenebis-acrylamide, and 0.1 g vinylphenylboronic acid was adjusted to pH 9by addition of aqueous NaOH, and subsequently polymerised. Silver wasadded to the resulting polymer using 0.2M AgNO₃ dissolved in 5% aceticacid. The hologram was then recorded in 20% methanol and developed in astandard developer; such developers are detailed in Practical Holographyby Graham Saxby, published by Prentice Hall.

Detection of glucose was made in the presence of a 0.1 M phosphatebuffer with 0.1 M NaCl solution at various pH values. The hologram wasplaced in 1 ml of buffer with a stirrer, and 20 ml of glucose in water(which had been left for 24 hours) was added to the buffer to a glucoseconcentration of 2 mM.

Readings were taken at 30 second intervals for a range of pH values. Thesystem was reversible at all pH values (including physiological pHvalues) tested.

For example, at pH 8.1, addition of glucose resulted in expansion of thepolymer, with most of the expansion taking place within 5 minutes ofglucose addition. This expansion was observed as a reduction in thewavelength of reflection, as shown in FIG. 1. After rinsing twice withbuffer, the hologram contracted, reverting to its original conformation;the wavelength returned to its original value, as shown in FIG. 2.

EXAMPLE 2 Detection of Oxygen Using “Vaska's Complex”

“Vaska's complex” was immobilised onto a plain HEMA hologram byevaporation from solution in chloroform (2 ml of 5 mg/ml solution). Thehologram was then sparged with oxygen in a nitrogen-saturated buffer (50mmol phosphate buffer, pH 7.0).

Upon sparging with oxygen, a small, reversible expansion occurred,resulting in an increase in wavelength of about 10 nm relative tocontrols. This wavelength shift is shown in FIG. 3. After spargingceased, a proportion of the bound oxygen molecules underwent the reversereaction and the holographic medium contracted slightly. This wasobserved as a slight reduction in wavelength.

EXAMPLE 3 Detection of Oxygen Using Oxygen-Binding Proteins

A holographic sensor was produced by immobilising haemoglobin onto aHEMA polymer by drying, followed by the addition of 1.5% acidifiedglutaraldehyde for 3 minutes. A holographic sensor comprisingimmobilised myoglobin was similarly produced.

The holograms were sparged with oxygen for approximately 5 minutes. Ascan be seen from FIG. 4, a response to changing oxygen concentration wasdisplayed by both haemoglobin and myoglobin containing holograms.

EXAMPLE 4 Detection of pH

Holograms comprising a polymer content of 8% methacrylic acid (MM) wereused to monitor the pH in Lactobacillus casei cultured in MRS broth. Asthe lactobacillus grew and produced lactic acid the hologram contractedrapidly in response to the fall in pH. This was observed as a shorteningof the peak wavelength of reflection of the hologram during thefermentation, as shown in FIG. 5.

When the peak wavelength of reflection of the hologram was compared withpH determined by a pH probe, a precise correlation was found, with less‘scatter’ associated with the hologram than the pH probe.

Similarly, holograms comprising 4% DMAEM (dimethylaminoethylmethacrylate)-HEMA were used to continuously monitor Escherichia colicultures, which also produce acid upon expansion. Peak wavelength ofreflectance of the holograms correlated closely with pH.

FIG. 6 shows the pH sensitivity of four different holographic sensors,wherein each sensor comprises a copolymer formed from HEMA and apH-sensitive comonomer. In this example, the pH-sensitive comonomersused were vinylimidazole, diethylaminomethacrylate, methacrylic acid andtrifluoropropenoic acid. The pH sensitivities are markedly different,showing how a polymer composition may be optimised for greatersensitivity over a specific pH range.

The following Examples 5-7 illustrate the invention using 3cyclodextrine (CD) derivatives to make sensors.

EXAMPLE 5 Cyclohexaamylose “Alpha CD”

1 g alpha CD (dried) dissolved in 5 ml dry DMF and 3 ml drytriethylamine. Flask then chilled in ice bath. 2.7 ml methacryloylchloride then added slowly to the stirred mixture in 100 microlitres(μl) increments over 10 minutes. The flask stoppered with CaCl₂ dryingtube, was left stirring at room temperature overnight. The mixture wasthen shaken with 100 ml toluene and the solvent was filtered off byBuchner funnel/water pump. The precipitate was then shaken with 100 ccacetone and again filtered and finally rinsed with 50 cc acetone anddried.

The methacrylated product was then stored in a dark bottle labelledalpha CD-M.

A “smart” polymer was then made as follows:

60 mg alpha CD-M was dissolved in 150 ul methanol.

The following were then added in order and each component dissolvedbefore adding the next.

5 mg DMPA

125 μl HEMA

10 μl EDMA

The solution was then coated on microscope slides and polymerized underUV light as previously described. (Alternatively the polymerizationcould be carried out thermally at around 60° C. for several hours if theDMPA was substituted by AIBN)

Holographic gratings were then made in the slides using the diffusionmethod as previously described.

EXAMPLE 6 Cycloheptaamylose or Beta-CD

1 g beta-CD (dried) dissolved in 5 ml dry DMF and 3 ml drytriethylamine. Flask then chilled in ice bath.

2.5 ml methacryloyl chloride then added slowly to the stirred mixture in100 μl increments over 10 minutes.

The flask stoppered with CaCl₂ drying tube, was left stirring at roomtemperature overnight.

The mixture was then shaken with 100 ml toluene and the solvent wasfiltered off by Buchner funnel water pump. The precipitate was thenshaken with 100 cc acetone and again filtered and finally rinsed with 60cc acetone and dried.

The methacrylated product was then stored in a dark bottle labelled betaCD-M.

A “smart” polymer was then made as follows:

60 mg beta CD-M was dissolved in 150 μl methanol.

The following were then added in order and each component dissolvedbefore adding the next.

5 mg DMPA

125 μl HEMA

10 μl EDMA

The solution was then coated on microscope slides and polymerized underUV light as previously described. (Alternatively the polymerizationcould be carried out thermally at around 60° C. for several hours if theDMPA was substituted by AIBN)

Holographic gratings were then made in the slides using the diffusionmethod as previously described.

EXAMPLE 7 Hydroxypyropyl Cylcooctaamylase or HP Gamma CD

1 g HP gamma CD (dried) dissolved in 5 ml dry DMF and 3 ml drytriethylamine.

Flask then chilled in ice bath.

2.5 ml methacryloyl chloride then added slowly to the stirred mixture in100 μl increments over 10 minutes.

The flask stoppered with CaCl₂ drying tube, was left stirring at roomtemperature overnight.

The mixture was then shaken with 100 ml toluene and the solvent wasfiltered off by Buchner funnel/water pump. The precipitate was thenshaken with 100 cc acetone and again filtered and finally rinsed with 50cc acetone and dried.

The methacrylated product was then stored in a dark bottle labelled HPgamma CD-M.

A “smart” polymer was then made as follows:

60 mg HP gamma CD-M was dissolved in 150 μl methanol.

The following were then added in order and each component dissolvedbefore adding the next.

5 mg DMPA

125 μl HEMA

10 μl EDMA

The solution was then coated on microscope slides and polymerized underUV light as previously described. (Alternatively the polymerizationcould be carried out thermally at around 60 C. for several hours if theDMPA was substituted by AIBN)

Holographic gratings were then made in the slides using the diffusionmethod as previously described.

The effect of CD cavity size with respect to analyte molecule size canbe seen in FIGS. 7 and 8.

EXAMPLE 8

This Example illustrates continuous monitoring of alcohol production bySaccharomyces cerevisiae using an alcohol sensor hologram.

Alcohol production by S. cerevisiae in grape juice was monitored over 24hours using an alcohol sensor hologram.

Exponential phase S. cerevisiae cells growing in white grape juice werespun down, re-suspended in 20% glycerol and frozen. Cell density wasdetermined by a viable count to be 8×10⁶ ml⁻¹.

0.5 ml of seed culture was added to 2.5 ml of white grape juice in acuvette and cultured at 30° C. for 24 hours. Alcohol (ethanol)production during this period was monitored using a polyHEMA alcoholsensor hologram, the peak wavelength of reflection at each time intervalcompared with a calibration produced using ethanol and grape juice (FIG.9). Ethanol production during 24 hours of culture is shown in FIG. 10.

1. A method for the detection of an analyte in a fluid, which comprisescontacting the fluid with a holographic element comprising a medium anda hologram disposed throughout the volume of the medium, wherein anoptical characteristic of the element changes as a result of a variationof a physical property occurring throughout the volume of the medium,wherein the variation arises as a result of interaction between themedium and the analyte, and wherein the reaction and the variation arereversible; and detecting any change of the optical characteristic. 2.The method according to claim 1, wherein the physical property is thesize of the medium.
 3. The method according to claim 1, wherein theoptical characteristic is the reflectance, refractance or absorbance ofthe holographic element.
 4. The method according to claim 1, wherein anychange of the optical characteristic is detected as a color change. 5.The method according to claim 1, wherein any change of the opticalcharacteristic is detected as an intensity change.
 6. The methodaccording to claim 1, wherein the analyte is glucose or lactate.
 7. Themethod according to claim 1, wherein the analyte is CO₂ or oxygen. 8.The method according to claim 1, wherein the contacting comprisespassing the fluid continuously over the element.
 9. The method accordingto claim 1, wherein the fluid is an optical fluid.
 10. A device for thedetection of an analyte in a fluid, which comprises a fluid conduithaving an inlet, an outlet, and a holographic element over which thefluid can flow, wherein the device also includes a window wherebynon-ionising radiation can irradiate the holographic element.
 11. Thedevice according to claim 10, wherein the holographic element comprisesa medium and a hologram disposed throughout the volume of the medium,wherein an optical characteristic of the element changes as a result ofa variation of a physical property occurring throughout the volume ofthe medium, wherein the variation arises as a result of interactionbetween the medium and the analyte, and wherein the reaction and thevariation are reversible.
 12. The device, according to claim 11, whereinthe physical property is the size of the medium.
 13. The device,according to claim 11, wherein the optical characteristic is thereflectance, refractance or absorbance of the holographic element. 14.The device, according to claim 11, wherein any change of the opticalcharacteristic is detected as an intensity change.
 15. The device,according to claim 11, wherein any change of the optical characteristicis detected as an intensity change.